ABSTRACT The combination of high frequencies of infection, high mortality rates, emergence of drug resistance, small number of therapeutic options and a depleted discovery pipeline has resulted in an urgent need for finding new antifungal molecules. Natural products, especially those produced by fungal species as natural antifungal weapons, have been and will continue to play important roles and provide leads for drug discovery. Realizing the immense potential of natural products as antifungal leads, and the prospects of discovering new structures through genome-guided methods, we are proposing a comprehensive and multidisciplinary project to investigate the biosynthesis of fungal-derived 2-pyridone alkaloids, which are complex natural products with potent and selective antifungal activities. We will be particularly focused on a diverse set of enzyme-catalyzed pericyclic reactions that give rise to the polycyclic scaffold important for biological activity. Uncovering these enzymes, followed by detailed investigation of the mechanisms and sequence-activity relationships, will improve our fundamental understanding of enzyme catalysis, and enable biosynthetic engineering approaches to generate natural product analogs, and expand genome mining leads for new antifungals. This multi-PI proposal bring together complementary expertise in three aspects of natural product research: biosynthesis, computational chemistry and synthesis. Together we will address three aims: 1) Investigate SAM-dependent pericyclase using leporin as a model system. 2) Investigate Alder-Ene and dephenylation reactions in pyridoxatin biosynthesis; and 3) Investigate biosynthesis of decalin-containing antifungal 2-pyridone compounds. Insights into the biosynthesis of these molecules will reveal fundamental insights into enzymes that catalyze pericyclic reactions, and provide leads for structure-guided natural product discovery.